Macromolecular protein or glycoprotein constituents derived from the testis have been shown to cause regression of the Mullerian ducts in the mammalian male embryo. Mullerian Inhibiting Substance is present in the postnatal testes of the rat, human, and calf. The postnatal calf testes has been the source for biochemical isolation and a specific constituent extracted from bovine testes has been characterized by density gradient sedimentation, column chromatography, gel electrophoresis, isoelectric focusing, amino acid analysis, and gas liquid chromatography. This investigation has been facilitated by the development of a reliable organ culture assay for Mullerian Inhibiting Substance capable of detecting activity both in testicular fragments and in soluble extracts. Antibody to the testicular extract which has been raised in rabbits and absorbed with fetal calf serum, demonstrates a single immunoprecipitant band against testicular extracts in purified fractions. Using gel electrophoresis and isoelectric focusing, single bands will be tested in the modified organ culture system for Mullerian Inhibiting Substance activity. An active band will be used to raise a specific antibody, and a radioimmunoassay developed. Ultrastructural studies have demonstrated a sequence of lysosome stimulation, autodigestion and autophagocytosis, leading to duct regression both in vivo and in vitro. Mesenchymal-epithelial, basement membrane, and extracellular matrix interactions will be studied during duct regression. The Sertoli cell will be studied during maximum Mullerian Inhibiting Substance production. Using an antibody to LHRH, our laboratory has shown that gonadatropins inhibit either testicular release or production of Mullerian Inhibiting Substance. FSH as a possible extrinsic modulator will be explored. Microcytotoxicity techniques will be used to demonstrate the sensitivity of Mullerian duct cells and tumors of Mullerian duct origin to Mullerian Inhibiting Substance. Mullerian Inhibiting Substance will be explored as a potential tumoricidal agent for tumor of Mullerian duct origin.